Project Summary Infectious diseases cause widespread sickness throughout the world each year and are the second leading cause of death, particularly in underdeveloped countries. And with the emergence of multi-drug resistance strains, the necessity for new, more effective, and more sustainable therapies is immediate. Included in these infectious diseases are the apicomplexa which includes Toxoplasma gondii and Plasmodium falciparum, the parasites causing toxoplasmosis and malaria, respectively. These parasites contain a unique plastid-like organelle called an apicoplast which contains four membranes and therefore have evolved a complex system for importing and exporting proteins across these membranes. These essential import/export machineries are ideal targets for novel antibiotics against these pathogens. Many of these translocon machineries are also conserved in other higher eukaryotic organelles such as chloroplasts and mitochondria, where a large majority of genes are nuclear encoded and therefore must be imported post-translationally. One such machinery is the conserved translocon of the outer membrane in chloroplasts (TOC) complex from Arabidopsis thaliana, a model system for studying chloroplast biology. The TOC complex consists of primarily three components, Toc33/34 and Toc159, both GTPases containing an N-terminal transmembrane helix anchoring them into the outer membrane, and Toc75, a 16- stranded ?-barrel membrane-spanning translocon. While mechanistic models have been put forth for how the TOC complex functions, they have remained largely unproven due to the lack of structural characterization, which is needed to stitch together all the pieces of the mechanistic puzzle. In our studies, we will use biophysical methods, X-ray crystallography, cryo-electron microscopy, and small-angle X-ray scattering to structurally and functionally characterize this specialized machinery. Our results will fill a longstanding gap in the field and will be essential for piecing together the mechanism for how the TOC complex functions in protein import in apicomplexa and chloroplasts.